This proposal is designed to optimize the monoclonal antibody-IFN fusion molecule (mAb-IFN) by introducing several point mutations in the IFN part of the molecule. Recently, we discovered that mAb- IFN fusion molecule is about 4 log units more potent at activating the type 1 IFN receptor (IFNAR) on cells which express the antibody target protein than it is at stimulating IFNAR on cells which do not express the antibody target protein. The mAb-IFN fusion molecule is about 2 log units less potent compared to non- fused IFN on cells which do not express the antibody target protein, and about 2 log units more potent than non-fused IFN at activating the IFNAR on cells which express the antibody target protein. The decrease in intrinsic potency of the mAb-IFN fusion molecule is likely due to steric hindrance, and the increase in activity on cells which express the target of the antibody is likely due to the antibody localizing the fused IFN to the cell surface, so that little or none ofthe intrinsic binding energy of IFN for its receptor has to be used to pay the cost of entropy loss incurred when the bound IFN is localized on the cell surface. Thus, the effective concentration of the fused IFN is much higher than its actual concentration. Thus, a way has been discovered to considerably increase the therapeutic index of IFN for treatment of cancer by fusing IFN to antibodies which bind selectively to cancer cells, since the antiproliferative an proapoptotic effects of IFN on cancer cells are desired for the therapeutic benefit, while stimulation of the receptor on non cancer cells causes the dose-limiting adverse effects of IFN. We and our collaborators at the UCLA will explore the effect of varying the intrinsic affinity of IFN for its receptor by introducing a series of mutations into IFN part of the fusion protein, then determining the effects of changes in affinity of IFN for its receptor on the potency difference of the fusion protein between stimulating IFNAR on cells which express the antibody target protein and those which do not. Based on the rationale provided above for the increase in potency of the fusion protein on cells which express the antibody target protein, in spite of a decrease in intrinsic potency of the fused IFN, we expect that further decreasing the intrinsic potency of the IFN in the fusion protein will further increase the potency difference between cells which do and do not express the target protein of the antibody, with the result of further increasing the therapeutic index of the fusion protein. We will test this prediction, and use our findings to drive selection of specific fusion molecules for development as cancer therapeutics. This proposal specifically focuses to optimize anti-CD20-IFN fusion molecule to treat human B cell malignancies. PUBLIC HEALTH RELEVANCE: IFN has potent anti-tumor activities; however its usage in the clinic has been restricted due to dose limiting toxicity. Monoclonal antibodies-IFN (mAb-IFN) fusion molecules, developed by ImmunGene, have demonstrated efficacy and safety in animal models of cancer which are resistant to the treatment with either the antibody alone, IFN alone, and to both in combination. Based on these data, we believe that mAb-IFN fusion molecules can be an effective treatment for a significant number of patients who do not respond to current cancer therapeutics. We propose to optimize the IFN part of the mAb-IFN fusion molecule to further increase the therapeutic index. An optimized therapeutic will overcome several limitations of existing targeted therapeutics, e.g. genetic (Fc?R polymorphism) and acquired drug resistance, lower dose - patients' convenience and cost effective.